Enhancers are critical cis-regulatory elements that regulate spatiotemporal gene expression and control cell fates.However,the identification of enhancers in native cellular contexts still remains a challenge.Here,we ...Enhancers are critical cis-regulatory elements that regulate spatiotemporal gene expression and control cell fates.However,the identification of enhancers in native cellular contexts still remains a challenge.Here,we develop an inducible CRISPR activation(CRISPRa)system by transgenic expression of doxycycline(Dox)-inducible dCas9-VPR in mouse embryonic stem cells(iVPR ESC).With this line,a simple introduction of specific guide RNAs targeting promoters or enhancers allows us to realize the effect of CRISPRa in an inducible,reversible,and Dox concentration-dependent manner.Taking advantage of this system,we induce tiled CRISPRa across genomic regions(105 kilobases)surrounding T(Brachyury),one of the key mesodermal development regulator genes.Moreover,we identify several CRISPRa-responsive elements with chromatin features of putative enhancers,including a region the homologous sequence in which humans harbors a body height risk variant.Genetic deletion of this region in ESC does affect subsequent T gene activation and osteogenic differentiation.Therefore,our inducible CRISPRa ESC line provides a convenient platform for high-throughput screens of putative enhancers.展开更多
基金This work was supported by the National key research and development program(2017YFA0504102 to X.W.)the National Natural Science Foundation of China(81772676,31970579 to X.W.,31900464 to T.L.)+1 种基金the Natural Science Foundation of Tianjin Municipal Science and Technology Commission(18JCJQJC48200 to X.W.)Tianjin Education Commission(2020ZD13 to X.W.,2018KJ075 to T.L.),Open grant from the Chinese Academy of Medical Sciences(157-Z20-04 to X.W.)。
文摘Enhancers are critical cis-regulatory elements that regulate spatiotemporal gene expression and control cell fates.However,the identification of enhancers in native cellular contexts still remains a challenge.Here,we develop an inducible CRISPR activation(CRISPRa)system by transgenic expression of doxycycline(Dox)-inducible dCas9-VPR in mouse embryonic stem cells(iVPR ESC).With this line,a simple introduction of specific guide RNAs targeting promoters or enhancers allows us to realize the effect of CRISPRa in an inducible,reversible,and Dox concentration-dependent manner.Taking advantage of this system,we induce tiled CRISPRa across genomic regions(105 kilobases)surrounding T(Brachyury),one of the key mesodermal development regulator genes.Moreover,we identify several CRISPRa-responsive elements with chromatin features of putative enhancers,including a region the homologous sequence in which humans harbors a body height risk variant.Genetic deletion of this region in ESC does affect subsequent T gene activation and osteogenic differentiation.Therefore,our inducible CRISPRa ESC line provides a convenient platform for high-throughput screens of putative enhancers.
